Polysaccharide pseudo-sponge

ABSTRACT

There is provided a photocrosslinked polysaccharide exhibiting a low swelling property and a high degradation ability in vivo while retaining a suitable strength. The polysaccharide pseudo-sponge is produced by a crosslinking reaction of a photoreactive polysaccharide obtained by introducing a photoreactive group into a polysaccharide, and exhibits a low swelling property and a blue dextran-low dyeaffinity which satisfy the following properties (I) and (II), respectively: (I) a swelling ratio of not more than 125% as calculated from the values measured by immersing a test specimen having a thickness of 1 mm, a length of 10 mm and a width of 10 mm, and a solvent content of 96% by weight, in water for injection at room temperature for 1 hour, according to the following formula: 
 
Swelling ratio={( S 2− S 1)/ S 1}×100 
wherein Si represents an area of the test specimen before the immersion, and S2 is an area of the test specimen after the immersion, in which the area is calculated from the length and width of the test specimen; and (II) an absorbance of not more than 0.15 at a wavelength of 620 nm as measured with respect to an aqueous solution containing 0.67% by weight of a polysaccharide which is prepared by immersing a test specimen having a thickness of 1 mm, a length of 20 mm and a width of 10 mm, and a solvent content of 96% by weight, in an aqueous solution containing 0.5 g/mL of blue dextran having a weight-average molecular weight of 2,000,000, and then subjecting the test specimen to water-washing and hydrolysis.

TECHNICAL FIELD

The present invention relates to a polysaccharide pseudo-sponge, andmore particularly, to a polysaccharide pseudo-sponge having combinedproperties of sponge and gel, which is produced by crosslinking reactionof photoreactive polysaccharide obtained by introducing a photoreactivegroup into polysaccharide.

BACKGROUND ART

Conventionally, there are known photoreactive polysaccharides obtainedby introducing a photoreactive group into polysaccharides, as well ascrosslinked polysaccharides obtained by crosslinking the photoreactivepolysaccharides by irradiation of light such as ultraviolet rays (forexample, Japanese Patent Application Laid-Open (KOKAI) Nos.6-73102(1994), 8-143604(1996), 9-87236(1997) and 2002-249501). Inaddition, there are also known gels (polysaccharide gels) or sponges(polysaccharide sponges) produced from such crosslinked polysaccharides.

The polysaccharide gels have been produced by irradiating a solution ofthe photoreactive polysaccharide with light such as ultraviolet rays forcrosslinking the photoreactive polysaccharide, and used, for example, asmedical materials such as antiadhesive materials for inhibiting adhesionof tissues of living organisms (for example, Japanese Patent ApplicationLaid-Open (TOKUHYO) No. 11-512778(1999)). Meanwhile, the abovepolysaccharide gels are obtained in the form of a solvated gel. Whenusing an aqueous solution of photoreactive polysaccharides as a rawmaterial, the resultant polysaccharide gels are in the form of ahydrogel due to hydration thereof. Such polysaccharide gels have athree-dimensional network structure, and therefore, are insoluble inwater, but swelled up until reaching an equilibrium condition thereof inwater.

On the other hand, the polysaccharide sponges are produced by freezing asolution of photoreactive polysaccharides and then irradiating thefrozen solution with light such as ultraviolet rays to crosslink thephotoreactive polysaccharides. In the production process, impuritiessuch as crosslinking agents are extremely easily removed from thereaction mixture, thereby enabling production of high-purity products(for example, WO 02/060971 A1). Meanwhile, the term “sponge” means aporous substance having closed cells or interconnecting cells.

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

A hydrogel of crosslinked glycosaminoglycan as an example ofpolysaccharide gels has an excellent degradation ability in vivo.However, the hydrogel tends to be readily split when molded into a gelsheet, etc., and must be handled with a great care. In addition, due toa high swelling property of the hydrogel, it may be difficult to preventdisplacement of the gel sheet when placed in vivo. Further, the problemconcerning easy split of the gel sheet tend to become more remarkabledue to the high swelling property, in particular, when used as anantiadhesive material in tissues of living organisms.

On the other hand, a sponge of the crosslinked glycosaminoglycan as anexample of the polysaccharide sponges exhibits a low swelling property,and therefore, tends to be hardly displaced when placed in vivo.However, the polysaccharide sponges have a porous structure and a lowdegradation ability in vivo, and therefore, tend to easily undergoinfiltration of cells, resulting in transformation of the sponges into afibrous structure. Accordingly, the polysaccharide sponges are usable invivo only in the specific limited applications such as scaffold forregeneration of tissues. Meanwhile, easy infiltration of cells into thepolysaccharide sponges is considered to be due to a porosity thereof.The porosity (mesh size) of the polysaccharide sponges may be estimatedfrom the degree of dyeing (dyeaffinity) with blue dextran which isgenerally considered to be hardly permeable into a network structure ofthe hydrogel. The polysaccharide sponges have a relatively largedyeaffinity.

The present invention has been conducted to solve the above problems. Anobject of the present invention is to provide a photocrosslinkedpolysaccharide having a novel physical structure, which can exhibit alow swelling property and a high degradation ability in vivo whileretaining a suitable strength thereof.

MEANS FOR SOLVING THE PROBLEM

As a result of the present inventor's earnest study for solving theabove problems and providing a photocrosslinked polysaccharide having anovel physical structure, which exhibits excellent combined propertiesof conventional crosslinked polysaccharide sponges and gels, it has beenfound that a specific polysaccharide pseudo-sponge obtained by impartingproperties of polysaccharide gels to polysaccharide sponges having alow-swelling property, can realize a novel physical structure capable ofovercoming defects of the conventional polysaccharide sponges. Thepresent invention has been attained on the basis of the above finding.That is, the present invention includes the following plural aspectsrelated to each other.

To accomplish the aim, in a first aspect of the present invention, thereis provided a polysaccharide pseudo-sponge produced by a crosslinkingreaction of a photoreactive polysaccharide obtained by introducing aphotoreactive group into a polysaccharide, the said polysaccharidepseudo-sponge exhibiting a low swelling property and a blue dextran-lowdyeaffinity which satisfy the following properties (I) and (II),respectively:

(I) a swelling ratio of not more than 125% as calculated from the valuesmeasured by immersing a test specimen having a thickness of 1 mm, alength of 10 mm and a width of 10 mm, and a solvent content of 96% byweight, in water for injection at room temperature for 1 hour, accordingto the following formula:Swelling ratio={(S2−S1)/S1}×100wherein S1 represents an area of the test specimen before the immersion,and S2 is an area of the test specimen after the immersion, in which thearea is calculated from the length and width of the test specimen; and

(II) an absorbance of not more than 0.15 at a wavelength of 620 nm asmeasured with respect to an aqueous solution containing 0.67% by weightof a polysaccharide which is prepared by immersing a test specimenhaving a thickness of 1 mm, a length of 20 mm and a width of 10 mm, anda solvent content of 96% by weight, in an aqueous solution containing0.5 g/mL of blue dextran having a weight-average molecular weight of2,000,000, and then subjecting the test specimen to water-washing andhydrolysis.

In a second aspect of the present invention, there is provided apolysaccharide pseudo-sponge which is produced by irradiating light to asolution of a photoreactive polysaccharide obtained by introducing aphotoreactive group into a polysaccharide to obtain a polysaccharide gelhaving a shape-retention property, freezing the obtained polysaccharidegel, and then irradiating light to the resultant frozen polysaccharidegel.

In a third aspect of the present invention, there is provided apolysaccharide pseudo-sponge which is produced by irradiating light to asolution of a photoreactive polysaccharide obtained by introducing aphotoreactive group into a polysaccharide to obtain a polysaccharide gelhaving a shape-retention property, freeze-drying the obtainedpolysaccharide gel, and then irradiating light to the resultantfreeze-dried polysaccharide gel.

In a fourth aspect of the present invention, there is provided a processfor producing a polysaccharide pseudo-sponge, comprising the steps ofirradiating light to a solution of a photoreactive polysaccharideobtained by introducing a photoreactive group into a polysaccharide toobtain a polysaccharide gel having a shape-retention property, freezingthe obtained polysaccharide gel, and then irradiating light to theresultant frozen polysaccharide gel.

In a fifth aspect of the present invention, there is provided a processfor producing a polysaccharide pseudo-sponge, comprising the steps ofirradiating light to a solution of a photoreactive polysaccharideobtained by introducing a photoreactive group into a polysaccharide toobtain a polysaccharide gel having a shape-retention property,freeze-drying the obtained polysaccharide gel, and then irradiatinglight to the resultant freeze-dried polysaccharide gel.

In a sixth aspect of the present invention, there is provided a medicalmaterial comprising the above polysaccharide pseudo-sponge.

EFFECT OF THE INVENTION

In accordance with the present invention, there are provided apolysaccharide pseudo-sponge having not only an excellentbiodegradability but also a good strength and a high barrier effectagainst adhesion in tissues, as well as a medical material using thepolysaccharide pseudo-sponge.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing a breaking strength of a polysaccharidepseudo-sponge 1 of the present invention.

FIG. 2 is a graph showing a blue dextran dyeaffinity of thepolysaccharide pseudo-sponge 1 of the present invention, in which a barwith a vertical stripe represents results obtained by a dipping method,whereas a bar with a horizontal stripe represents results obtained by asoaking method.

FIG. 3 is a graph showing a swelling property of the polysaccharidepseudo-sponge 1 of the present invention.

FIG. 4 is a graph showing an enzymatic degradation ability of thepolysaccharide pseudo-sponge 1 of the present invention.

FIG. 5 is a graph showing percentages of residual blue dextran andresidual polysaccharide pseudo-sponge as measured for a period, in whicha blue dextran-containing polysaccharide pseudo-sponge is embedded in anabdominal cavity of a rat.

FIG. 6 is an enlarged view of the surface of a polysaccharidepseudo-sponge 2 according to the present invention.

FIG. 7 is an enlarged view of a section of a polysaccharidepseudo-sponge 2 according to the present invention.

FIG. 8 is an enlarged view of the surface of a crosslinked hyaluronicacid gel produced in Production Example 2(2).

FIG. 9 is an enlarged view of a section of the crosslinked hyaluronicacid gel produced in Production Example 2(2).

FIG. 10 is an enlarged view of the surface of a crosslinked hyaluronicacid sponge produced in Production Example 3(2).

FIG. 11 is an enlarged view of a section of the crosslinked hyaluronicacid sponge produced in Production Example 3(2).

PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention is described in detail below. For the convenienceof explanation, the process for producing a polysaccharide pseudo-spongeaccording to the present invention is first explained. In the presentinvention, similarly to the conventional photocrosslinkedpolysaccharides, a photoreactive polysaccharide obtained by introducinga photoreactive group into a polysaccharide is used as a raw material.The photoreactive polysaccharide can be produced by reacting thepolysaccharide with a compound (photoreactive substance) capable ofundergoing a photo-dimerization reaction or a photo-polymerizationreaction therewith upon irradiation with light.

Examples of the polysaccharide may include homoglycan, heteroglycan andderivatives thereof. The homoglycan is a polysaccharide constituted froma single kind of monosaccharide solely. Examples of the homoglycan mayinclude glucans such as amylose and cellulose; mannan; glycuronans suchas pectic acid and alginic acid; polyglycosamines such as chitin andcolominic acid; polygalactosamines; or the like. Among thesehomoglycans, preferred are glucans, and more preferred is cellulose.

Specific examples of the derivatives of homoglycans may includecarboxymethylated derivatives such as carboxymethyl cellulose,hydroxymethylated derivatives such as hydroxymethyl cellulose, andde-acetylated derivatives such as chitosan. Among these derivatives ofhomoglycans, preferred are water-soluble derivatives, more preferred arecarboxymethylated derivatives, in particular, carboxymethyl cellulose,and hydroxymethylated derivatives, in particular, hydroxymethylcellulose, and still more preferred are carboxymethylated homoglycans.

The heteroglycan is a polysaccharide constituted from two kinds ofsugars. Among the heteroglycans, preferred are glycosaminoglycan orderivatives thereof. Specific examples of the glycosaminoglycan mayinclude hyaluronic acid, chondroitin, chondroitin sulfate, dermatansulfate, heparin, heparan sulfate, keratan sulfate or the like. Specificexamples of the glycosaminoglycan derivatives may include sulfatedderivatives such as sulfated hyaluronic acid and chondroitinpolysulfate; de-sulfated derivatives such as de-sulfated heparin; andoxidation-reduction derivatives such as periodic acidoxidation-reduction heparin and periodic acid oxidation-reductionde-sulfated heparin (Japanese Patent Application Laid-Open (KOKAI) No.11-310602(1999)). Examples of the de-sulfated heparin may include6-position de-sulfated heparin (WO 00/06608 A1) and 2-positionde-sulfated heparin (Japanese Patent Application Laid-Open (KOKAI) No.2003-113090).

The weight-average molecular weight of the polysaccharide used in thepresent invention varies depending upon kinds thereof. When hyaluronicacid is used as the polysaccharide, the weight-average molecular weightof the hyaluronic acid is usually 200,000 to 3,000,000, preferably300,000 to 2,000,000, more preferably 400,000 to 1,200,000, and whenother sugars are used as the polysaccharide, the weight-averagemolecular weight thereof is usually 4,000 to 2,500,000.

The photoreactive group may be a residue of a compound (photoreactivesubstance) having a photoreactive crosslinking group or a photoreactiveunsaturated bond, which is capable of undergoing a photo-dimerizationreaction or a photo-polymerization reaction by irradiation with lightsuch as ultraviolet rays to form a crosslinked structure. Meanwhile, thekind of photoreactive group is not particularly limited as long as itcan undergo polymerization or dimerization upon irradiation with light,and unless glycoside bond of the polysaccharide is cut or broken byintroducing the photoreactive group thereinto.

Examples of the photoreactive substance may include cinnamic acid,substituted cinnamic acids such as aminocinnamic acid formed byreplacing any hydrogen atom bonded to a benzene ring of cinnamic acidwith an amino group, preferably p-aminocinnamic acid; acrylic acid;maleic acid; fumaric acid; furyl-acrylic acid; thiophene-acrylic acid;cinnamylidene-acetic acid; sorbic acid; thymine; coumarin; andderivatives thereof. Among these photoreactive substances, cinnamicacid, substituted cinnamic acids and derivatives thereof are preferredin the consideration of safety.

In addition, a spacer may be bonded to the photoreactive substance toenhance a photoreactivity thereof and facilitate the photo-crosslinkingreaction or the reaction for introducing the photoreactive group intothe polysaccharide. The spacer is preferably a divalent or polyvalentfunctional compound having a chain-like or cyclic hydrocarbon residuehaving 2 to 18 carbon atoms. For example, in the case where cinnamicacid is used as the photoreactive substance, the spacer is preferablyamino alcohol having 2 to 8 carbon atoms. In this case, the aminoalcohol is ester-bonded or amide-bonded to a carboxyl group of thecinnamic acid. More preferably, the spacer is n-amino propanol orn-amino butanol.

The photoreactive polysaccharide may be produced by known methods asdescribed, for example, in Japanese Patent Application Laid-Open (KOKAI)Nos. 6-73102(1994) and 8-143604(1996), Japanese Patent ApplicationLaid-Open (TOKUHYO) No. 11-512778(1999), etc.

In the process of the present invention, the photoreactivepolysaccharide is used in the form of a solution thereof. Here, the“solution” means a liquid in which the photoreactive polysaccharide isdissolved or uniformly dispersed. The solvent used for preparing thephotoreactive polysaccharide solution is not particularly limited aslong as the solution can be frozen or freeze-dried after irradiatinglight thereto while keeping the photoreactive polysaccharide dissolvedor dispersed therein. The solvent is preferably an aqueous solvent.Examples of the aqueous solvent may include a phosphate buffered saline,distilled water, water for injection, etc.

The concentration of the photoreactive polysaccharide in the solutionmay be appropriately determined according to the relationship between amolecular weight of the polysaccharide and a degree of substitution ofthe photoreactive group, and is usually 0.1 to 10% by weight. Forexample, when the photoreactive group is introduced at a degree ofsubstitution of 0.1 to 15% into hyaluronic acid having a weight-averagemolecular weight of 400,000 to 1,200,000, the concentration of thephotoreactive polysaccharide in the solution is preferably 0.5 to 8% byweight.

Meanwhile, the “degree of substitution of the photoreactive group” meansthe value by percentage which is calculated from a ratio of a number ofmoles of the “photoreactive group introduced” (number of thephotoreactive substances introduced) to a number of moles of “functionalgroups into which the photoreactive group can be introduced” which iscontained in the polysaccharide. The functional group of thepolysaccharide into which the photoreactive group can be introduced, mayvary depending upon kind of the photoreactive group or spacer. When acarboxyl group contained in the photoreactive group or spacer is to bebonded to the polysaccharide, examples of the functional group of thepolysaccharide may include an amino group and a hydroxyl group. Whereas,when an amino or hydroxyl group contained in the photoreactive group orspacer is to be bonded to the polysaccharide, examples of the functionalgroup of the polysaccharide may include a carboxyl group.

Before subjecting the photoreactive polysaccharide solution tocrosslinking reaction by irradiation with light, substances other thanthe photoreactive polysaccharide and solvent such as unreactedphotoreactive substance, impurities and foreign materials are preferablyremoved therefrom, thereby enhancing a purity of the resultantpolysaccharide pseudo-sponge of the present invention to such an extentthat the sponge is usable in medical applications such as medicalequipments. The removal of the impurities and foreign materials, etc.,which are contained in the solution may be conducted, for example, byordinary methods such as dialysis, filtration and centrifugalseparation. The photoreactive polysaccharide is usually produced in theform of an aqueous solvent solution. Therefore, the photoreactivesubstance which is unreacted with the polysaccharide can be extremelyeasily removed from the solution. Such a removal treatment is veryadvantageous, in particular, for producing the polysaccharidepseudo-sponge of the present invention which is difficult to clean.

Next, the photoreactive polysaccharide solution is irradiated with lightto obtain a polysaccharide gel having a shape-retention property. Then,the obtained polysaccharide gel is frozen or freeze-dried, and theresultant frozen or freeze-dried product of the polysaccharide gel isfurther irradiated with light.

The light irradiation is preferably conducted through a container usedfor retaining a shape of the solution. In particular, such a containeris preferably used upon irradiating light to the frozen polysaccharidegel. The shape of the container is usually determined in theconsideration of the shape of the finally obtained polysaccharidepseudo-sponge of the present invention. In this case, for example, whenusing a photoreactive polysaccharide produced by using a photoreactivesubstance having an unsaturated double bond which is capable ofundergoing a crosslinking reaction upon absorption of ultraviolet rays,such as cinnamoyl group (residue of cinnamic acid), since ultravioletrays tend to be absorbed by water used as a solvent in the crosslinkingreaction, the light irradiation is preferably controlled such that anoptical path length of the ultraviolet rays is not more than 1 cm. It isrequired that the material of the container is selected from thosematerials incapable of absorbing light required for the crosslinkingreaction of the photoreactive polysaccharide, i.e., capable of allowingsuch a light to penetrate therethrough. Examples of the material of thecontainer which is suitably used in the crosslinking reaction usingultraviolet rays may include polymer compounds such as polypropylenehaving a low ultraviolet absorptivity, glass such as quartz glass andhard glass, etc. Meanwhile, when irradiating light to the freeze-driedproduct, the use of the container is not necessarily required, and lightmay be directly irradiated onto the freeze-dried product.

The light irradiated is not particularly limited as long as thephotoreactive substance undergoes the reaction such as polymerizationand dimerization. Examples of the light irradiated which is involved inthe present invention may include visible light, ultraviolet rays,infrared rays, electron beams and radiation rays. Among these light andrays, preferred are visible light and ultraviolet rays, and morepreferred are ultraviolet rays. The wavelength of the light used ispreferably 180 to 650 nm. For example, in the case where cinnamic acidis used as the photoreactive substance, there may be suitably usedultraviolet rays having a wavelength of 260 to 350 nm. Suitableirradiation conditions for obtaining the polysaccharide gel having ashape-retention property may be conveniently determined by conductingpreliminary experiments.

The freezing conditions used for freezing the polysaccharide gelobtained by irradiating the photoreactive polysaccharide solution withlight, are not particularly limited as long as the polysaccharide gel issuitably frozen thereunder, and the polysaccharide gel can be frozen byconventionally known methods ordinarily used for production ofpolysaccharide sponges. For example, the polysaccharide gel may berapidly frozen using an ultra-low temperature substance such as liquidnitrogen, or a cooling medium cooled below a freezing point of thepolysaccharide gel such as ethanol. Alternatively, the polysaccharidegel may be relatively slowly frozen by cooling the gel using generaldomestic refrigerators. Meanwhile, the freezing treatment may be usuallysuccessively conducted after the step of producing the polysaccharidegel. The polysaccharide gel accommodated in the container may bedirectly cooled. Also, when the polysaccharide gel obtained byirradiating the photoreactive polysaccharide solution with light, isfreeze-dried, there may be used ordinary freeze-drying methods. Forexample, there may be used the method of freezing the polysaccharide gelat −20° C., and then freeze-drying the frozen product at roomtemperature under reduced pressure, e.g., under 1 pascal (Pa).

It is known that the frozen or freeze-dried product undergoes acrosslinking reaction with a remarkably small amount of light energy ascompared to that required for crosslinking reaction of a solution.Therefore, it is economically advantageous that production of thepolysaccharide gel having a shape-retention property before the freezingor freeze-drying treatment is conducted by irradiating a minimumnecessary amount of light, and a sufficient light irradiation forattaining the aimed crosslinking ratio is conducted after the freezingor freeze-drying treatment.

The amount of light irradiated may appropriately vary depending uponaimed applications of the polysaccharide pseudo-sponge of the presentinvention. Here, the “amount of light” is calculated from a product ofan “illuminance per unit area” and an “irradiation time”. For example,in the case where aminopropyl cinnamate obtained by bonding aminopropanol to cinnamic acid and hyaluronic acid is used as thephotoreactive substance and polysaccharide, respectively, the followingprocedure may be conducted in order to obtain the polysaccharidepseudo-sponge of the present invention which exhibits a relatively highmechanical strength.

First, an aqueous solution containing, for example, 4% by weight ofphotoreactive hyaluronic acid having 8% as a degree of substitution ofphotoreactive group is filled in a container capable of allowing thesolution to be irradiated with light from both sides thereof, and lightis irradiated to the solution from both sides of the container in anamount of 50 J/cm² per one side thereof (measuring wavelength: 280 nm)to obtain a polysaccharide gel having a shape-retention property. Next,the thus obtained polysaccharide gel is frozen, and light is irradiatedto the frozen gel from both sides thereof in an amount of 100 to 250mJ/cm² per one side thereof (measuring wavelength: 280 nm), therebyobtaining a photocrosslinked hyaluronic acid pseudo-sponge. Meanwhile,when using a 3 kw high-pressure mercury lamp, the light irradiation onlyfrom one side is possible. In such a case, in order to compensateunevenness of light irradiation, the container filled with the aqueoussolution containing a photoreactive hyaluronic acid or the frozen gel isturned every irradiation of light, thereby allowing the frozen gel to beirradiated with light from both sides thereof.

The total amount of light irradiated, for example, upon producing thepolysaccharide gel may be not less than about 1,000 mJ/cm², and ispreferably 100,000 mJ/cm². Further, in order to obtain the pseudo-spongeof the present invention by irradiating light to the frozenpolysaccharide gel, the total amount of light to be irradiated to thefrozen polysaccharide gel may be not less than 10 mJ/cm², and ispreferably 500 mJ/cm². In the case where the polysaccharide gel obtainedby irradiating the photoreactive polysaccharide solution with light isfreeze-dried, since the light transmittance of the freeze-driedpolysaccharide gel is lower than that of the frozen product, the totalamount of light to be irradiated is usually not less than 500 mJ/cm²,preferably not less than 5 J/cm², more preferably not less than 10J/cm².

Meanwhile, the amount of light irradiated may be measured, for example,using an illuminance meter “UV-M10” (manufactured by ORC ManufacturingCo., Ltd.), but may also be measured by ordinary apparatuses capable ofirradiating a similar amount of light.

In addition, in the production method of the present invention, thephotoreactive polysaccharide solution may also comprise any material(additive) exhibiting a miscibility with an aqueous solvent which isselected from the group consisting of alcohols, surfactants andchelating agents. For example, after dissolving the photoreactivepolysaccharide and the above additive in the aqueous solvent to preparea photoreactive polysaccharide solution, the resultant solution may beirradiated with light to obtain a polysaccharide gel having ashape-retention property. Then, after freezing or freeze-drying theobtained polysaccharide gel, the thus obtained frozen or freeze-driedproduct may be irradiated with light to obtain the polysaccharidepseudo-sponge of the present invention.

Meanwhile, the additive having a miscibility with the aqueous solventwhich is selected from the group consisting of alcohols, surfactants andchelating agents, must be selected from those materials incapable ofinhibiting functions or effect of the polysaccharide pseudo-sponge ofthe present invention. Examples of the alcohols having a miscibilitywith the aqueous solvent may include those alcohols, in particular,polyethyleneglycols, which are represented by the following generalformula (1):R—OH  (1)wherein R is any group selected from the group consisting of thefollowing groups (a) to (e):

(a) a chain-like alkyl group having 1 to 10 carbon atoms;

(b) a branched alkyl group having 3 to 10 carbon atoms;

(c) —CH₂—(CHOH)₁—CH₂OH, wherein 1 is an integer of 0 to 5;

wherein m is an integer of 3 to 5; and

(e) —(CH₂CH₂O)_(n)—H, wherein n is an integer of 3 to 70.

Examples of the chain-like alkyl group having 1 to 10 carbon atoms mayinclude methyl, ethyl or the like. Examples of the branched alkyl grouphaving 3 to 10 carbon atoms may include isopropyl, t-butyl or the like.

Examples of the above alcohols may include lower alcohols, polyhydricalcohols and sugar alcohols.

Examples of the lower alcohols may include alcohols having 1 to 10carbon atoms, preferably 1 to 8 carbon atoms. Specific examples of thelower alcohols may include methanol, ethanol, isopropanol, t-butylalcohol or the like. The polyhydric alcohols are alcohols containing 2or more hydroxyl groups, preferably 3 or more hydroxyl groups, in amolecule thereof. Specific examples of the polyhydric alcohols mayinclude ethyleneglycol and glycerol. Among these polyhydric alcohols,preferred is ethyleneglycol. Also, the sugar alcohols usable in thepresent invention may be either chain-like sugar alcohols or cyclicsugar alcohols, preferably chain-like alcohols. Examples of the sugaralcohols may include inositol, mannitol, xylitol, sorbitol or the like.Among these sugar alcohols, preferred are mannitol, xylitol andsorbitol, and more preferred are mannitol and sorbitol.

As the surfactants, there are preferably used nonionic surfactant andanionic surfactants. Examples of the nonionic surfactants may includepolyethyleneglycol (PEG). Examples of the anionic surfactants mayinclude salts of alkylsulfuric acids, preferably sodium dodecylsulfate.

Examples of the chelating agents may include oxycarboxylic acids such ascitric acid, and polyaminocarboxylic acids such as edetic acids, e.g.,salts of ethylenediaminetetraacetic acid (EDTA).

The additive having a miscibility with the aqueous solvent which isselected from the group consisting of the above alcohols, surfactantsand chelating agents, may be suitably selected according to propertiesor applications of the obtained polysaccharide pseudo-sponge. Forexample, the polysaccharide pseudo-sponge produced according to theprocess of the present invention using citric acid as the additive canbe enhanced in strength and adhesion property when formed into a sheetshape. Also, the polysaccharide pseudo-sponge obtained by adding any ofglycerol and polyethyleneglycol 400 (PEG 400) thereto can be enhanced inflexibility when formed into a sheet shape. Further, as mentioned below,in the case where the polysaccharide pseudo-sponge of the presentinvention is used as a medical material for sustained release of drugsby previously impregnating the drugs thereinto, for example, in order toimpregnate the polysaccharide pseudo-sponge with lipid-soluble drugs,glycerol and PEG 400 may be suitably selected as the additive, and EDTAmay be suitably selected as the additive for impregnating thepolysaccharide pseudo-sponge with a basic fibroblast growth factor.

Next, the polysaccharide pseudo-sponge of the present invention isexplained. As described above, the polysaccharide pseudo-sponge of thepresent invention is produced by subjecting the photoreactivepolysaccharide obtained by introducing a photoreactive group into apolysaccharide to crosslinking reaction. The polysaccharidepseudo-sponge of the present invention is characterized by exhibiting alow swelling property and a blue dextran-low dyeaffinity which satisfythe following properties (I) and (II), respectively:

(I) a swelling ratio of not more than 125% as calculated from the valuesmeasured by immersing a test specimen having a thickness of 1 mm, alength of 10 mm and a width of 10 mm, and a solvent content of 96% byweight, in water for injection at room temperature for 1 hour, accordingto the following formula:Swelling ratio={(S2−S1)/S1}×100wherein S1 represents an area of the test specimen before the immersion,and S2 is an area of the test specimen after the immersion, in which thearea is calculated from the length and width of the test specimen; and

(II) an absorbance of not more than 0.15 at a wavelength of 620 nm asmeasured with respect to an aqueous solution containing 0.67% by weightof a polysaccharide which is prepared by immersing a test specimenhaving a thickness of 1 mm, a length of 20 mm and a width of 10 mm, anda solvent content of 96% by weight, in an aqueous solution containing0.5 g/mL of blue dextran having a weight-average molecular weight of2,000,000, and then subjecting the test specimen to water-washing andhydrolysis.

The polysaccharide pseudo-sponge of the present invention which isproduced by irradiating a photoreactive polysaccharide solution withlight to obtain a polysaccharide gel, freezing the polysaccharide gel,and then irradiating the thus obtained frozen polysaccharide gel withlight, exhibits the same solvation condition as that of conventionalpolysaccharide gels, i.e., the polysaccharide pseudo-sponge of thepresent invention may be produced in a solvent-containing state. When anaqueous solution of the photoreactive polysaccharide is used as the rawmaterial, the polysaccharide pseudo-sponge may be produced in a hydrousstate. Thus, when the aqueous solution of the photoreactivepolysaccharide is used as the photoreactive polysaccharide solution uponproducing the polysaccharide pseudo-sponge of the present invention, theabove solvent content means a water content thereof. That is, forexample, when using a 4 wt % aqueous solution of cinnamicacid-introduced hyaluronic acid, there is obtained a polysaccharidepseudo-sponge containing 96% by weight of water, namely, the solventcontent (water content) thereof is 96% by weight. Meanwhile, apolysaccharide pseudo-sponge obtained by drying the above hydrouspolysaccharide pseudo-sponge is also involved in the scope of thepresent invention.

On the other hand, the polysaccharide pseudo-sponge of the presentinvention which is produced by irradiating the photoreactivepolysaccharide solution with light to obtain a polysaccharide gel,freeze-drying the polysaccharide gel and then irradiating the thusobtained freeze-dried polysaccharide gel with light, contains no solventsuch as water unlike the above solvent-containing polysaccharidepseudo-sponge. However, the solvent-free polysaccharide pseudo-spongemay be immersed in a solvent such as water to obtain thesolvent-containing polysaccharide pseudo-sponge. For example, a testspecimen used for examining properties of the polysaccharidepseudo-sponge (water content: 96% by weight) may be prepared byimmersing a water-free sample in distilled water used in an amount 24times a weight of the sample to cause the sample to absorb water, andthen cut the water-impregnated sample into a desired size. In addition,there may also be used such a test specimen which is produced bypreviously molding the polysaccharide gel into a desired size beforefreeze-drying. Meanwhile, the test for low swelling property may beconducted by comparing a size of a test specimen before freeze-drying,which is prepared by previously molding the polysaccharide gel into asize of 1 cm in length×1 cm in width×1 mm in thickness, with a size ofthe same test specimen which is allowed to absorb a sufficient amount ofdistilled water after freeze-drying.

As described above, the low swelling property in water is inherent topolysaccharide sponges. That is, although the polysaccharide gel isswelled up by solvation, the polysaccharide sponges undergosubstantially no swelling (this property is not largely influenced by acrosslinking ratio thereof). The polysaccharide pseudo-sponge of thepresent invention exhibits a low swelling property, i.e., a swellingratio of not more than 125% as measured by the above swelling test (I),which is similar to that of the polysaccharide sponges. The swellingratio of the polysaccharide pseudo-sponge of the present invention ispreferably not more than 100%, more preferably not more than 70%. Themuch lower swelling property of the polysaccharide pseudo-sponge can beachieved, for example, by enhancing a crosslinking ratio thereof.

In the above blue dextran-dyeaffinity test (II), water for injection isusually used for preparing an aqueous blue dextran solution. Also, thetest specimen of the polysaccharide pseudo-sponge is hydrolyzed for 1hour by adding 1 mL of 1 mol/L NaOH thereto. Upon the hydrolysis, awhole portion of the test specimen is dissolved, thereby preparing apolysaccharide solution containing the dye used for tinting the testspecimen. The absorbance of the polysaccharide solution may be measuredusing a spectrophotometer.

In the present invention, as the method for tinting the test specimenwith blue dextran, the following two methods may be used for ensuringformation of the tinted test specimen. One of the methods is a so-calledsoaking method in which the test specimen is immersed in 1 mL of a bluedextran solution for 1 hour, taken out from the solution, and thenlightly rinsed in water for injection; and the other method is aso-called dipping method in which after repeating the operation ofsuspending and dipping the test specimen in a blue dextran solution 10times, the operation of suspending and dipping the test specimen inwater for injection is repeated 10 times. In the former method, the testspecimen is tinted or dyed over a sufficient period of time, whereas inthe latter method, the test specimen is tinted or dyed while preventingswelling thereof.

Meanwhile, since the polysaccharide sponge inherently has a high bluedextran dyeaffinity, it is not essentially expectable that thepolysaccharide sponge can exhibit a low dyeaffinity with blue dextran.Rather, since it is generally considered that blue dextran cannot bepenetrated into a network structure of a hydrogel, the low dyeaffinitywith blue dextran is a property exhibited by the polysaccharide gel.However, the polysaccharide pseudo-sponge of the present inventionexhibits such a low dyeaffinity that an absorbance thereof is not morethan 0.15 as measured by the above method shown in the below-mentionedExamples. Such a low dyeaffinity of the polysaccharide pseudo-sponge ofthe present invention is considered to be due to polysaccharide gel-likeproperties thereof. The absorbance of the polysaccharide pseudo-spongeaccording to the present invention as measured by the dyeaffinity testis preferably not more than 0.10, more preferably not more than 0.05. Amuch lower dyeaffinity of the polysaccharide pseudo-sponge can beachieved by enhancing a crosslinking ratio thereof. Meanwhile, the bluedextran dyeaffinity is considered to have a correlation with a barriereffect of preventing adhesion of tissues or cells in vivo, andtherefore, is usable as an index for estimating the barrier effect.Thus, the low blue dextran dyeaffinity of the polysaccharidepseudo-sponge of the present invention suggests a high barrier effect ofthe polysaccharide pseudo-sponge for inhibiting the adhesion of tissues,etc.

The polysaccharide pseudo-sponge of the present invention preferably hasa breaking strength of not less than 200 g as measured by piercing andbreaking a test specimen having a thickness of 1 mm, a length of 60 mmand a width of 25 mm, and a solvent content of 96% by weight, with a12.7 mm-diameter spherical probe at 24° C. and a piercing speed of 1mm/s by using a texture analyzer. Such a high breaking strength of thepolysaccharide pseudo-sponge cannot be achieved even by conventionalpolysaccharide sponges as shown in the below-mentioned Examples, andtherefore, is one of unique properties exhibited by the polysaccharidepseudo-sponge of the present invention. The breaking strength of thepolysaccharide pseudo-sponge obtained by the method of irradiating thefrozen polysaccharide gel with light is preferably not less than 250 g,more preferably not less than 300 g, whereas the breaking strength ofthe polysaccharide pseudo-sponge obtained by the method of irradiatingthe freeze-dried polysaccharide gel with light is preferably not lessthan 210 g, more preferably not less than 220 g. The higher breakingstrength of the polysaccharide pseudo-sponge can be achieved byenhancing a crosslinking ratio thereof.

The polysaccharide pseudo-sponge of the present invention preferably hasan enzymatic degradation time of not more than 1300 minute as measuredat 50° C. by subjecting a test specimen having a thickness of 1 mm, alength of 20 mm and a width of 10 mm, and a solvent content of 96% byweight, to a polysaccharide degrading enzyme (e.g., hyaluronidase) in areaction mixture containing 1 mL of a 5 mmol/L phosphate bufferedsaline, 0.2 mL of a 1 mol/L acetate buffer solution and 0.2 mL of a 5TRU(Turbidity Reducing Unit)/mL the enzyme solution. The enzymaticdegradation time of the polysaccharide pseudo-sponge of the presentinvention is preferably not more than 1,250 min, more preferably notmore than 1,200 min, still more preferably not more than 1,000 min. Theabove-specified enzymatic degradation time of the polysaccharidepseudo-sponge can be achieved, for example, by controlling acrosslinking ratio thereof.

The polysaccharide pseudo-sponge of the present invention ischaracterized by exhibiting combined properties of polysaccharide spongeand polysaccharide gel as is apparent from the above low swellingproperty and the low blue dextran dyeaffinity thereof. The reason whythe polysaccharide pseudo-sponge of the present invention have suchunique properties, is considered as follows, though not clearlydetermined.

That is, as described above, the frozen or freeze-dried product tends toreadily undergo crosslinking reaction with a remarkably small amount oflight energy as compared to that required for crosslinking reaction of asolution. In addition, the frozen product is more readily subjected tocrosslinking reaction as the freezing temperature thereof is lowered. Itis considered that such a high crosslinking efficiency is due to phaseseparation of the photoreactive polysaccharide solution into a solventand a solute thereof, enhanced orientation (crystallinity) of thephotoreactive polysaccharide molecules, lowering of thermal motion, etc.Meanwhile, the polysaccharide pseudo-sponge of the present invention isobtained by two-stage crosslinking reaction including a first-stagecrosslinking reaction conducted in a solution state and a second-stagecrosslinking reaction conducted in a frozen or freeze-dried state. Inthis case, for example, the following phenomena are suggested.

(a) In the first-stage slow crosslinking reaction, a small amount of athree-dimensional network structure is formed. Then, in the second-stagecrosslinking reaction conducted in a frozen or freeze-dried state, thereis formed such a dense structure in which the photoreactivepolysaccharide molecules are arranged within the three-dimensionalnetwork structure. As a result, there is formed a composite structure inwhich coarse and dense portions are three-dimensionally arranged, forexample, like a reinforced concrete construction.

(b) In the first-stage slow crosslinking reaction, the three-dimensionalnetwork structure is totally formed, and after thus enhancingorientation of the photoreactive polysaccharide molecules, thesecond-stage crosslinking reaction is conducted in a frozen orfreeze-dried state. As a result, as compared to the case whereorientation of the photoreactive polysaccharide molecules is made onlyin the second-stage crosslinking reaction in a frozen or freeze-driedstate, there can be formed such a high orientation structure in whichthe three-dimensional orientation of the photoreactive polysaccharidemolecules is further enhanced.

In any case, it is considered that the polysaccharide pseudo-sponge ofthe present invention has a different structure from those ofconventional polysaccharide sponges and polysaccharide gels owing to theabove unique properties thereof. Further, it is considered that thepolysaccharide pseudo-sponge of the present invention has a highbreaking strength which is unachievable even by the conventionalpolysaccharide sponges owing to the above composite structure (a) and/orhigh orientation structure (b).

Next, the medical material of the present invention is explained. Themedical material of the present invention is characterized by comprisingthe above polysaccharide pseudo-sponge of the present invention. In theprocess for production of the polysaccharide pseudo-sponge of thepresent invention, impurities or foreign materials are readily removedtherefrom as described above. In particular, the polysaccharides such asglycosaminoglycan are present in vivo. Therefore, it is considered thatthe polysaccharide pseudo-sponge of the present invention exhibits ahigh safety when used in vivo, thereby enabling the polysaccharidepseudo-sponge to be used as a medical material. Specific examples of themedical material may include an antiadhesive material used in tissues ofliving organisms, for example, post-operative antiadhesive materialsused upon operation.

Meanwhile, in the case where the antiadhesive material has a highswelling property, the antiadhesive material tends to be displaced fromthe aimed region where adhesion of tissues is to be inhibited, resultingin poor antiadhesive effect. For example, a sheet-like antiadhesivematerial having a high swelling property tends to be deteriorated instrength upon swelling, resulting in easy split and breakage uponapplication of an external pressure thereto. As a result, there tends tobe caused such a significant problem that the material no longer acts asa barrier for keeping tissues apart from each other. On the other hand,since the polysaccharide pseudo-sponge of the present invention has anextremely low swelling property, the antiadhesive material obtainedtherefrom can be hardly displaced from the aimed region. In addition,the antiadhesive material of the present invention undergoessubstantially no split and breakage even when used in the form of asheet, and therefore, is very useful as a barrier material. Since thepolysaccharide pseudo-sponge of the present invention is readilydegraded in vivo, there is caused no problem that the antiadhesivematerial stays in vivo for a too long period of time. Further, due tothe above low blue dextran dyeaffinity, the polysaccharide pseudo-spongeof the present invention is considered to exhibit a good resistance toinfiltration of cells. Such a property is suitable, in particular, asantiadhesive materials.

Also, examples of the other medical material may include a base materialfor sustained release of drugs. More specifically, the polysaccharidepseudo-sponge of the present invention, which exhibits an excellentdegradation ability in vivo can be used as a base material for sustainedrelease of drugs by previously impregnating drugs thereinto. Such amedical material for sustained release of drugs may be produced bypreviously mixing drugs in the photoreactive polysaccharide solutionupon producing the polysaccharide pseudo-sponge of the presentinvention. Alternatively, the base material for sustained release ofdrugs may also be produced by simply impregnating the polysaccharidepseudo-sponge of the present invention with drugs.

Examples of the drugs which can be contained in the polysaccharidepseudo-sponge of the present invention may include non-steroidalanti-inflammatory drugs such as indomethacin, mefenamic acid,acemethacin, alclofenac, ibuprofen, thiaramide hydrochloride, fenbufen,mepirizole and salicylic acid; anti-malignant tumor drugs such asmethotrexate, fluorouracil, vincristine sulfate, mitomycin C,actinomycin C and daunorubicin hydrochloride; antiulcer drugs such asaceglutamide aluminum, L-glutamine,P-(trans-4-aminomethylcyclohexanecarbonyl)-phenyl propionic acidhydrochloride, cetraxate hydrochloride, sulpiride, gefarnate,cimetidine, ranitidine and famotidine; enzyme preparations such aschymotrypsin, streptokinaze, lysozyme chloride, bromelain, urokinase andtissue plasminogen activator; antihypertensive drugs such as clonidinehydrochloride, bunitrolol hydrochloride, prazosin hydrochloride,captopril, bethanidine sulfate, metoprolol tartrate and methyldopa;urogenital drugs such as flavoxate hydrochloride; antithrombus drugssuch as heparin, heparan sulfate, thrombomodulin, dicumarol andwaffarin; antiarteriosclerosis drugs such as clofibrate, simfibrate,elastase and nicomol; circulatory drugs such as nicardipinehydrochloride, nimodipine hydrochloride, cytochrome C and tocopherolnicotinate; steroid drugs such as hydrocortisone, prednisolone,dexamethasone and betamethasone; wound therapeutic accelerators such asgrowth factors, heparin derivatives and collagen; as well asphysiologically active polypeptide, hormone agents, anti-tuberculosisdrugs, styptics, anti-diabetes therapeutic drugs, vasodilators,anti-arrhythmia drugs, cardiac drugs, anti-allergic drugs,anti-dipressant drugs, anti-epilepsy drugs, muscular relaxants,antitussive/expectorant drugs, antibiotics, etc.

Further, the polysaccharide pseudo-sponge of the present invention hassuch a property of preventing cells, etc., from being infiltratedthereinto, and therefore, can be suitably used as a base material ofculture medium for cells or tissues.

EXAMPLES

The present invention is described in more detail below by Examples, butthe Examples are only illustrative and not intended to limit the scopeof the present invention. Meanwhile, the definitions of technical termsand measuring methods used in the Examples are as follows.

(1) Degree of Substitution of Photoreactive Group

The degree of substitution of a photoreactive group in the case wherethe photoreactive group is introduced into a carboxyl group ofglycosaminoglycan as a polysaccharide, means a percentage of the numberof photoreactive groups introduced per a repeating disaccharide unit.The amount of glycosaminoglycan required for calculation of the degreeof substitution of a photoreactive group was measured by a carbazolemethod using a calibration curve thereof, and the amount of cinnamicacid when using cinnamic acid or a substituted cinnamic acid as aphotoreactive substance was measured by an absorbance measuring method(measuring wavelength: 269 nm) using a calibration curve thereof.

(2) Crosslinking Ratio

The crosslinking ratio was determined as follows. That is, 1 g of asample material to be measured was hydrolyzed with 1 mL of a 1 mol/Lsodium hydroxide aqueous solution for 1 hour. Then, after acidifying theobtained solution, photoreactive group-derived substances (monomers,dimers, etc.) were extracted with ethyl acetate, and the resultantextract was analyzed by a high-pressure liquid chromatography (HPLC) tomeasure an amount of the dimers by the method using a calibration curvethereof. The crosslinking ratio was expressed by a percentage of molesof the photoreactive groups converted into dimers to moles ofphotoreactive groups introduced into the polysaccharide.

Production Example 1 (Production of Photoreactive Hyaluronic Acid)

A mixed solution containing 250 mL of water and 375 mL of dioxane wasadded under stirring to 500 g of a 1 wt % aqueous solution of sodiumhyaluronate (comb-derived product, produced by Seikagaku Co., Ltd.;weight-average molecular weight: 900,000). The resultant mixed solutionwas successively mixed with a solution prepared by dissolving 860 mg ofN-hydroxysuccinimide in 2 mL of water (0.6 equivalent/hyaluronic aciddisaccharide unit (mol/mol)), a solution prepared by dissolving 717 mgof 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDCl/HCl) in 2 mL of water (0.3 equivalent/hyaluronic acid disaccharideunit (mol/mol)) and a solution prepared by dissolving 903 mg ofaminopropyl cinnamate hydrochloride (HCl.H₂N(CH₂)₃OCO—CH═CH—Ph, whereinPh represents a phenyl group) in 2 mL of water (0.3equivalent/hyaluronic acid disaccharide unit (mol/mol)) at roomtemperature, and stirred for 2.5 hours. Then, the resultant reactionsolution was mixed with a solution prepared by dissolving 2.5 g ofsodium hydrogencarbonate in 50 mL of water, followed by stirring for oneday, and further mixed with 30 g of sodium chloride. 2 L of ethanol wasadded to the obtained reaction solution to precipitate a solid. The thusprecipitated solid was washed with a mixed solvent composed of ethanoland water (weight ratio: 80:20) two times and then with ethanol twotimes, and dried at room temperature over night, thereby obtaining 5.24g of a white solid (3-aminopropyl cinnamate-introduced hyaluronic acid:also referred to as “cinnamic acid-introduced hyaluronic acid”). It wasconfirmed that the degree of substitution of cinnamic acid per repeatinghyaluronic acid disaccharide unit was 8.2%. Meanwhile, aminopropylcinnamate-introduced alginic acid and aminopropyl cinnamate-introducedcarboxymethyl cellulose were respectively produced by the same method asdescribed above.

Production Example 2 (Production of Crosslinked Hyaluronic Acid Gel)

(1) The cinnamic acid-introduced hyaluronic acid obtained in ProductionExample 1 was dissolved in water for injection to prepare a 4 wt %aqueous solution thereof. The resultant solution was poured into a moldmade of a reinforced glass plate which had a mold cavity of 6 cm×2.5cm×1 mm (in thickness), and then irradiated with ultraviolet rays usinga 3 kW metal halide lamp under water-cooling for 15 min for each surfacethereof, thereby obtaining a transparent sheet-shaped gel (watercontent: 96% by weight). It was confirmed that the crosslinking ratio ofthe obtained gel was 30%.

(2) The same procedure as defined in the above (1) was conducted exceptfor using a cinnamic acid-introduced hyaluronic acid having 4.6% as adegree of substitution, produced by the same method as defined inProduction Example 1, thereby obtaining a transparent sheet-shaped gel.It was confirmed that the crosslinking ratio of the obtained gel was13.6%. Meanwhile, the light irradiation was conducted using a 3 kWhigh-pressure mercury lamp such that the total amount of lightirradiated was 100 J/cm².

Production Example 3 (Production of Crosslinked Hyaluronic Acid Sponge)

(1) The cinnamic acid-introduced hyaluronic acid obtained in ProductionExample 1 was dissolved in water for injection to prepare a 4 wt %aqueous solution thereof. The resultant solution was poured into a moldmade of a reinforced glass plate which had a mold cavity of 6 cm×2.5cm×1 mm (in thickness), and then frozen at −20° C. The resultant frozenproduct was irradiated with ultraviolet rays in an amount of 2,000mJ/cm² using a 800 w high-pressure mercury lamp from both surfacesthereof, thereby obtaining an opaque white sheet-shaped sponge (watercontent: 96% by weight). It was confirmed that the crosslinking ratio ofthe obtained sponge was 33%.

(2) The same procedure as defined in the above (1) was conducted exceptfor using a cinnamic acid-introduced hyaluronic acid having 4.6% as adegree of substitution, produced by the same method as defined inProduction Example 1, thereby obtaining an opaque white sheet-shapedsponge. It was confirmed that the crosslinking ratio of the obtainedsponge was 16.2%. Meanwhile, the light irradiation was conducted using a800 W high-pressure mercury lamp such that the total amount of lightirradiated was 4 J/cm².

Example 1 (Production of Polysaccharide Pseudo-Sponge 1 of the PresentInvention)

As a filling container for a photoreactive polysaccharide solution,there was used a mold made of a reinforced glass plate which had a moldcavity of 6 cm in length×2.5 cm in width×1 mm in thickness. First, thecinnamic acid-introduced hyaluronic acid obtained in Production Example1 was dissolved in water for injection to prepare a 4 wt % aqueoussolution thereof. The resultant solution was poured into the above moldand then irradiated with ultraviolet rays in an amount of 50 J/cm² foreach surface thereof using a 3 kW metal halide lamp under water-coolingsuch that the total amount of light irradiated was 100 J/cm², andthereafter frozen at −20° C. The resultant frozen product was irradiatedwith ultraviolet rays in an amount of 100 mJ/cm² using a 800 Whigh-pressure mercury lamp from both surfaces thereof, thereby obtaininga translucent sheet-shaped polysaccharide pseudo-sponge 1 of the presentinvention (water content: 96% by weight). It was confirmed that thecrosslinking ratio of the obtained polysaccharide pseudo-sponge 1 was33%.

Example 2 (Production of Polysaccharide Pseudo-Sponge 2 of the PresentInvention)

The same procedure as defined in Example 1 was conducted except forusing a cinnamic acid-introduced hyaluronic acid having 4.6% as a degreeof substitution, produced by the same method as defined in ProductionExample 1, thereby obtaining a translucent sheet-shaped polysaccharidepseudo-sponge 2 of the present invention. It was confirmed that thecrosslinking ratio of the obtained polysaccharide pseudo-sponge 2 was17.0%. Meanwhile, the light irradiation after being frozen at −20° C.was conducted using a 800 W high-pressure mercury lamp such that thetotal amount of light irradiated was 1 J/cm².

<Breaking Strength Test>

The polysaccharide pseudo-sponge 1 of the present invention was testedby the method described in the present specification to measure abreaking strength thereof. In the test, there was used a textureanalyzer “TA-XT2” (manufactured by Stable Micro Systems Co., Ltd.).Further, as control samples, the crosslinked hyaluronic acid gelproduced in Production Example 2(1) and the crosslinked hyaluronic acidsponge produced in Production Example 3(1) were subjected to the samemeasurement as described above. The results of the measurement are shownin FIG. 1. As a result, it was confirmed that the polysaccharidepseudo-sponge 1 of the present invention had a breaking strength ofabout 420 g which was not less than 8 times that of the crosslinkedhyaluronic acid gel and slightly less than 4 times that of thecrosslinked hyaluronic acid sponge.

<Blue Dextran Dyeaffinity Test>

The polysaccharide pseudo-sponge 1 of the present invention wassubjected to dyeing test by the method (dipping method and soakingmethod) described in the present specification. Further, as controlsamples, the crosslinked hyaluronic acid gel produced in ProductionExample 2(1) and the crosslinked hyaluronic acid sponge produced inProduction Example 3(2) were subjected to the same measurement asdescribed above. The results of the measurement are shown in FIG. 2. InFIG. 2, the bar with a vertical stripe represents results by a dippingmethod, whereas the bar with a horizontal stripe represents results by asoaking method. It was confirmed that the polysaccharide pseudo-sponge 1of the present invention had such a dyeaffinity that an absorbancethereof was 0.02 which was about 1/7 time that of the crosslinkedhyaluronic acid gel and about 1/10 time that of the crosslinkedhyaluronic acid sponge. Meanwhile, it was considered that theunexpectedly high dyeaffinity of the gel was due to not penetration ofblue dextran into the gel but absorption of blue dextran onto thesurface of the gel.

<Swelling Test>

The polysaccharide pseudo-sponge 1 of the present invention wassubjected to swelling test by the method described in the presentspecification. Further, as control samples, the crosslinked hyaluronicacid gel produced in Production Example 2(1) and the crosslinkedhyaluronic acid sponge produced in Production Example 3(1) weresubjected to the same measurement as described above. The results of themeasurement are shown in FIG. 3. In FIG. 3, the area ratio means A2/A1wherein A1 represents an area of the test specimen before immersing inwater for injection, and A2 represents an area of the test specimenafter immersing in water for injection. Meanwhile, the area of the testspecimen means an area calculated from the length and width thereof. Asa result, it was confirmed that the polysaccharide pseudo-sponge 1 ofthe present invention had a swelling ratio of about 20% (area ratio:1.2), the crosslinked hyaluronic acid sponge underwent no swelling(swelling ratio: 0%; area ratio: 1.0), and the crosslinked hyaluronicacid gel had a swelling ratio of 240% (area ratio: 3.4).

<Enzymatic Degradation Ability Test>

The polysaccharide pseudo-sponge 1 of the present invention wassubjected to degradation ability test by the method described in thepresent specification. Further, as control samples, the crosslinkedhyaluronic acid gel produced in Production Example 2(1) and thecrosslinked hyaluronic acid sponge produced in Production Example 3(1)were subjected to the same measurement as described above. The resultsof the measurement are shown in FIG. 4. As a result, it was confirmedthat the polysaccharide pseudo-sponge 1 of the present inventionexhibited a degradation time of about 500 min which was 1.6 times thatof the crosslinked hyaluronic acid gel. On the other hand, thecrosslinked hyaluronic acid sponge required a degradation time 4.8 timesthat of crosslinked hyaluronic acid gel. Due to this fact, it wasconfirmed that the polysaccharide pseudo-sponge 1 of the presentinvention had an adequate enzymatic degradation ability.

Example 3 (Production of Polysaccharide Pseudo-Sponge 3 of the PresentInvention)

The cinnamic acid-introduced hyaluronic acid having 8.2% as a degree ofsubstitution, produced by the same method as defined in ProductionExample 1 was dissolved in water for injection to prepare a 4 wt %aqueous solution thereof. Next, the resultant solution was poured intothe same container as used in Example 1 and then irradiated withultraviolet rays in an amount of 50 J/cm² for each surface thereof usinga 3 kW metal halide lamp under water-cooling such that the total amountof light irradiated was 100 J/cm², and thereafter frozen at −20° C. Theresultant frozen product was taken out of the container, andfreeze-dried at room temperature. The thus obtained freeze-dried productwas irradiated with ultraviolet rays using a 800 W high-pressure mercurylamp from both surfaces thereof such that the total amount of lightirradiated was 5 J/cm², thereby obtaining a polysaccharide pseudo-sponge3. It was confirmed that the crosslinking ratio of the obtainedpolysaccharide pseudo-sponge 3 was 21.5%.

Various properties of the polysaccharide pseudo-sponge 3 were measuredby the same methods as used for the polysaccharide pseudo-sponge 1. As aresult, it was confirmed that the polysaccharide pseudo-sponge 3exhibited a breaking strength of 229.4 g, an absorbance of 0.021 asmeasured by dyeaffinity test using a dipping method and a soakingmethod, and a swelling ratio of 50%.

<Study on Sustained Drug-Releasability of Polysaccharide Pseudo-Spongeof the Present Invention>

The cinnamic acid-introduced hyaluronic acid having 8.2% as a degree ofsubstitution, produced by the same method as defined in ProductionExample 1 was dissolved in water for injection to prepare a 4 wt %aqueous solution thereof. Then, blue dextran having a molecular weightof 2,000,000 as a model substance for drugs was added to the thusobtained solution such that the concentration of blue dextran in thesolution was 10 mg/mL, and intimately mixed with each other. Theresultant solution was poured into a mold having a mold cavity of 5.5 cmin length×3.5 cm in width×0.5 mm in thickness, and then irradiated withultraviolet rays in an amount of 50 J/cm² for each surface thereof usinga 3 kW metal halide lamp under water-cooling such that the total amountof light irradiated was 100 J/cm², and thereafter frozen at −20° C. Theresultant frozen product was irradiated with ultraviolet rays whilebeing kept in a frozen state using a 800 W high-pressure mercury lampfrom both surfaces thereof such that the total amount of lightirradiated was 500 mJ/cm², thereby obtaining a blue dextran-containingpolysaccharide pseudo-sponge (hereinafter occasionally referred to as“BD-containing polysaccharide pseudo-sponge”). It was confirmed that thecrosslinking ratio of the obtained BD-containing polysaccharidepseudo-sponge was 20%. Five sheets of the BD-containing polysaccharidepseudo-sponge were prepared.

Using four rats, a sheet of the thus obtained BD-containingpolysaccharide pseudo-sponge was placed within a middle-incisedabdominal cavity of each rat, and the respective sheets of theBD-containing polysaccharide pseudo-sponge were recovered from an insideof the abdominal cavity after passage of each of one week, two weeks,three weeks and four weeks to measure a residual amount of blue dextranin the BD-containing polysaccharide pseudo-sponge as well as a residualamount of the polysaccharide pseudo-sponge itself therein.

The measurement of residual amount of the blue dextran was conducted bymeasuring a 620 nm absorbance of a solution prepared by hydrolyzing therecovered BD-containing polysaccharide pseudo-sponge with 50 mL of a 1NNaOH solution at room temperature for 1 hour, using a spectrophotometer.The residual amount of the blue dextran at each recovery time wasdetermined as a ratio based on an amount (100) of blue dextran containedin the BD-containing polysaccharide pseudo-sponge before being placedwithin the abdominal cavity.

The residual amount of the polysaccharide pseudo-sponge was measured byquantitative determination of an uronic acid content in the solutionused above for measuring the residual amount of the blue dextran usingcarbazole method. The residual amount of the polysaccharidepseudo-sponge at each recovery time was determined as a ratio of thethus measured uronic acid content to an uronic acid content (100) in theBD-containing polysaccharide pseudo-sponge before being placed withinthe abdominal cavity.

The results of measurements of residual percentages of the blue dextranand polysaccharide pseudo-sponge with respect to each imbedding periodof the sponge within the abdominal cavity of rats, are shown in FIG. 5.

From the results shown in FIG. 5, it was confirmed that even afterpassage of 4 weeks from the placement of the sponge within the abdominalcavity, the residual percentage of the blue dextran was about 22% andthe residual percentage of the polysaccharide pseudo-sponge was 70%.Although release of the blue dextran which was considered to be due toburst of the sponge, was recognized after one week from dosage of thesponge, since the residual amount of the blue dextran was subsequentlydecreased with reduction in residual amount of the polysaccharidepseudo-sponge, it was suggested that the blue dextran was released alongwith degradation of the polysaccharide pseudo-sponge. As a result, itwas confirmed that the polysaccharide pseudo-sponge in which a drug issimply mixed could continuously release the drug over a period of onemonth or longer without causing any chemical bond therebetween.Therefore, it is considered that the polysaccharide pseudo-sponge of thepresent invention can be suitably used as a base material for sustainedrelease of drugs.

<Observation Using Scanning Electron Microscope>

The polysaccharide pseudo-sponge 2 of the present invention was observedby a scanning electron microscope. Upon the observation by a scanningelectron microscope, there was used a freeze-dried product of thepolysaccharide pseudo-sponge 2. FIG. 6 shows an enlarged view(photograph as substitute for drawing) of the surface of thepolysaccharide pseudo-sponge 2 of the present invention, and FIG. 7shows an enlarged view (photograph as substitute for drawing) of thesection of the polysaccharide pseudo-sponge 2 of the present invention.Also, the crosslinked hyaluronic acid gel produced in Production Example2(2) and the crosslinked hyaluronic acid sponge produced in ProductionExample 3(2) were similarly subjected to the observation by a scanningelectron microscope. FIG. 8 shows an enlarged view (photograph assubstitute for drawing) of the surface of the crosslinked hyaluronicacid gel produced in Production Example 2(2); FIG. 9 shows an enlargedview (photograph as substitute for drawing) of the section of thecrosslinked hyaluronic acid gel produced in Production Example 2(2);FIG. 10 shows an enlarged view (photograph as substitute for drawing) ofthe surface of the crosslinked hyaluronic acid sponge produced inProduction Example 3(2); and FIG. 11 shows an enlarged view (photographas substitute for drawing) of the section of the crosslinked hyaluronicacid sponge produced in Production Example 3(2).

From the results of the observation by a scanning electron microscope,the reason why the polysaccharide pseudo-sponge of the present inventionexhibits a high strength is suggested as follows. That is, thepolysaccharide pseudo-sponge has a steric structure constituted fromplanes, whereas the sponge has a structure constituted from “columns(lines)”. Since the pseudo-sponge and the sponge have substantially nodifference in pore size therebetween, it is readily suggested that thestrength of the pseudo-sponge constituted from planes is higher thanthat of the sponge constituted from lines. In addition, thepolysaccharide pseudo-sponge has a considerably uniform wall surface.The smooth surface is usually improved in strength as compared to arough surface. Further, the polysaccharide pseudo-sponge is constitutedfrom a continuity of bags like closed cells, whereas the sponge has astructure composed of interconnected pores, and the gel is barred buthas a bag structure.

It is considered that the above difference in structure between thepolysaccharide pseudo-sponge and the sponge and gel reflects thedifference in blue dextran dyeaffinity therebetween. That is, thepseudo-sponge has a smooth surface as if the surface thereof is coveredwith a thin film, so that the blue dextran solution is readily flowedthereover, resulting in low dyeaffinity thereof. On the other hand, thereason why the gel exhibited a certain dyeaffinity notwithstanding thegel is usually known as a substance through which blue dextran particleshaving a weight-average molecular weight of 2,000,000 cannot bepenetrated, is considered to be that the blue dextran particles wereadsorbed on the rough surface of the gel.

Further, the swelling property of the gel is considered as follows. Thatis, since a wall surface of the gel is of a pleated shape, it issuggested that the gel has a high-order folded structure. Therefore, itis considered that the gel exhibits a swelling property due to strainwithin the high-order structure as well as margin in the expandingdirection thereof.

Example 4 (Production of Polysaccharide Pseudo-Sponge of the PresentInvention Using Cinnamic Acid-Introduced Alginic Acid)

One gram of photoreactive alginic acid obtained by introducingaminopropyl cinnamate into 4% of whole carboxyl groups of sodiumalginate (produced by Wako Junyaku Kogyo Co., Ltd.) was dissolved in 25mL of water for injection to prepare a 4 wt % photoreactive alginic acidaqueous solution. 1 mL of the resultant aqueous solution was filled andsealed in a high-density polypropylene pack such that a thickness of theaqueous solution filled in the pack was 1 mm, irradiated with light inan amount of 2,500 mJ/cm² using a 800 W high-pressure mercury lamp, andthen frozen in a dry ice/ethanol bath at −40° C. Next, the resultantfrozen product was further irradiated with light in an amount of 250mJ/cm² using a high-pressure mercury lamp while being kept in a frozenstate, thereby obtaining a crosslinked alginic acid pseudo-sponge.

Example 5 (Production of Polysaccharide Pseudo-Sponge of the PresentInvention Using Cinnamic Acid-Introduced Carboxymethyl Cellulose)

One gram of photoreactive carboxymethyl cellulose obtained byintroducing aminopropyl cinnamate into about 10% of whole carboxylgroups of sodium carboxymethyl cellulose (produced by Nakari-Tesc Co.,Ltd.) was dissolved in 25 mL of water for injection to prepare a 4 wt %photoreactive carboxymethyl cellulose aqueous solution. 1 mL of theresultant aqueous solution was filled and sealed in a high-densitypolypropylene pack such that a thickness of the aqueous solution filledin the pack was 1 mm, irradiated with light in an amount of 2,500 mJ/cm²using a 800 W high-pressure mercury lamp, and then frozen in a dryice/ethanol bath at −40° C. Next, the resultant frozen product wasfurther irradiated with light in an amount of 250 mJ/cm² using ahigh-pressure mercury lamp while being kept in a frozen state, therebyobtaining a crosslinked carboxymethyl cellulose pseudo-sponge.

1. A polysaccharide pseudo-sponge produced by a crosslinking reaction ofa photoreactive polysaccharide obtained by introducing a photoreactivegroup into a polysaccharide, said polysaccharide pseudo-spongeexhibiting a low swelling property and a blue dextran-low dyeaffinitywhich satisfy the following properties (I) and (II), respectively: (I) aswelling ratio of not more than 125% as calculated from the valuesmeasured by immersing a test specimen having a thickness of 1 mm, alength of 10 mm and a width of 10 mm, and a solvent content of 96% byweight, in water for injection at room temperature for 1 hour, accordingto the following formula:Swelling ratio={(S2−S1)/S1}×100 wherein Si represents an area of thetest specimen before the immersion, and S2 is an area of the testspecimen after the immersion, in which the area is calculated from thelength and width of the test specimen; and (II) an absorbance of notmore than 0.15 at a wavelength of 620 nm as measured with respect to anaqueous solution containing 0.67% by weight of a polysaccharide which isprepared by immersing a test specimen having a thickness of 1 mm, alength of 20 mm and a width of 10 mm, and a solvent content of 96% byweight, in an aqueous solution containing 0.5 g/mL of blue dextranhaving a weight-average molecular weight of 2,000,000, and thensubjecting the test specimen to water-washing and hydrolysis
 2. Apolysaccharide pseudo-sponge which is produced by irradiating light to asolution of a photoreactive polysaccharide obtained by introducing aphotoreactive group into a polysaccharide to obtain a polysaccharide gelhaving a shape-retention property, freezing the obtained polysaccharidegel, and then irradiating light to the resultant frozen polysaccharidegel.
 3. A polysaccharide pseudo-sponge which is produced by irradiatinglight to a solution of a photoreactive polysaccharide obtained byintroducing a photoreactive group into a polysaccharide to obtain apolysaccharide gel having a shape-retention property, freeze-drying theobtained polysaccharide gel, and then irradiating light to the resultantfreeze-dried polysaccharide gel.
 4. The polysaccharide pseudo-spongeaccording to claim 2, wherein the solution of the photoreactivepolysaccharide obtained by introducing a photoreactive group into apolysaccharide, further comprises an aqueous solvent-miscible substanceselected from the group consisting of alcohols, surfactants andchelating agents.
 5. The polysaccharide pseudo-sponge according to claim1, wherein the light irradiated has a wavelength of 180 to 650 nm. 6.The polysaccharide pseudo-sponge according to claim 1, wherein acrosslinking ratio of the polysaccharide pseudo-sponge is not less than1%.
 7. The polysaccharide pseudo-sponge according to claim 1, wherein abreaking strength of the polysaccharide pseudo-sponge is not less than200 g as measured by piercing and breaking a test specimen having athickness of 1 mm, a length of 60 mm and a width of 25 mm, and a solventcontent of 96% by weight, with a 12.7 mm-diameter spherical probe at 24°C. and a piercing speed of 1 mm/s by using a texture analyzer.
 8. Thepolysaccharide pseudo-sponge according to claim 1, wherein thepolysaccharide is homoglycan, heteroglycan or a derivative thereof. 9.The polysaccharide pseudo-sponge according to claim 8, wherein thehomoglycan is glucan.
 10. The polysaccharide pseudo-sponge according toclaim 8, wherein the heteroglycan is glycosaminoglycan.
 11. Thepolysaccharide pseudo-sponge according to claim 10, wherein theglycosaminoglycan is hyaluronic acid.
 12. The polysaccharidepseudo-sponge according to claim 1, wherein an enzymatic degradationtime of the polysaccharide pseudo-sponge is not more than 1300 minute asmeasured by subjecting a test specimen having a thickness of 1 mm, alength of 20 mm and a width of 10 mm, and a solvent content of 96% byweight, to a polysaccharide degrading enzyme in a reaction mixturecontaining 1 mL of a 5 mmol/L phosphate buffered saline, 0.2 mL of a 1mol/L acetate buffer solution and 0.2 mL of a 5TRU (Turbidity ReducingUnit)/mL the enzyme solution at 50° C.
 13. The polysaccharidepseudo-sponge according to claim 1, wherein the photoreactive group is aresidue of a compound formed by ester-bonding or amide bonding aminoalcohol to a carboxyl group of cinnamic acid.
 14. A process forproducing a polysaccharide pseudo-sponge, comprising the steps ofirradiating light to a solution of a photoreactive polysaccharideobtained by introducing a photoreactive group into a polysaccharide toobtain a polysaccharide gel having a shape-retention property, freezingthe obtained polysaccharide gel, and then irradiating light to theresultant frozen polysaccharide gel.
 15. A process for producing apolysaccharide pseudo-sponge, comprising the steps of irradiating lightto a solution of a photoreactive polysaccharide obtained by introducinga photoreactive group into a polysaccharide to obtain a polysaccharidegel having a shape-retention property, freeze-drying the obtainedpolysaccharide gel, and then irradiating light to the resultantfreeze-dried polysaccharide gel.
 16. The process according to claim 14,wherein the polysaccharide is homoglycan, heteroglycan or a derivativethereof.
 17. The process according to claim 16, wherein the homoglycanis glucan.
 18. The process according to claim 16, wherein theheteroglycan is glycosaminoglycan.
 19. The process according to claim18, wherein the glycosaminoglycan is hyaluronic acid.
 20. A medicalmaterial comprising the polysaccharide pseudo-sponge as defined inclaim
 1. 21. The medical material according to claim 20, which is usedas an antiadhesive material.
 22. The medical material according to claim20, which is used as a base material for sustained release of drug.